Two human mast cell types were identified by immunohistochemical techniques in skin, lung, and small intestine. One type contains the neutral proteases, tryptase and chymotryptic proteinase, and is termed the TC mast cell. The second type contains only tryptase and is termed the T mast cell. Both types are fixed better by Carnoy's fluid than by formalin. (4,5,(9)(10)(11).The presence of distinct chymotryptic neutral proteases in rat mucosal and connective tissue mast cells is a useful criterion for designation of a rat mast cell to a particular type (12, 13). For example, the finding of rat mucosal mast cell chymotryptic protease in a tumor cell line previously considered to be a rat basophil leukemia tumor (RBL-1) indicated that this tumor may be a rat mucosal mast cell leukemia tumor (14). In dispersed human lung mast cells, the major neutral protease is tryptase (15-17). Tryptase is a specific marker for all mast cells in human lung, small bowel, and skin (17-19), including formalin-fixable and -nonfixable mast cells. A second neutral protease, termed human skin chymotryptic proteinase, has been purified from skin (20) and localized to mast cells in skin and lung (21,22). The present study uses murine monoclonal anti-tryptase and affinity-purified rabbit polyclonal anti-skin chymotryptic proteinase antibodies in a double indirect immunoenzymatic technique to demonstrate two mast cell types in human skin, lung, and small bowel, based on distinct protease compositions. MATERIALS AND METHODSMaterials. Peroxidase-conjugated goat anti-rabbit IgG (Cooper Biomedical, Malvern, PA), alkaline phosphataseconjugated goat anti-mouse IgG (Boehringer Mannheim), peroxidase-conjugated goat anti-mouse IgG (Bio-Rad), 3,3-diaminobenzidine tetrahydrochloride (Electron Microscopy Sciences, Fort Washington, PA), 30% H202 (Fisher), and naphthol AS-MX phosphoric acid and fast blue RR (Sigma) were obtained as indicated. Murine monoclonal IgG2b,K anti-tryptase, termed G5 (anti-T), and a negative control IgG2b,K, termed MPC-11 (17), as well as polyclonal rabbit IgG anti-human skin chymotryptic proteinase, termed C8 (anti-C), and IgG from nonimmunized rabbits (21) were purified as described previously. Fresh surgical or autopsy tissues were fixed in Carnoy's fluid (60% methanol/30% chloroform/10% glacial acetic acid) or in neutral buffered formalin (10% formalin in 0.08 M sodium phosphate, pH 7.4) and 5-,gm-thick sections were prepared. Macroscopically normal appearing areas of skin, lung, and small bowel were used. In addition, mast cells were enzymatically dispersed from human lung and cytocentrifuge preparations were obtained as described (17).Single Indirect Immunohistochemistry. Fixed sections were dewaxed in xylene over three 5-min periods. Endogenous peroxidase was blocked with 0.6% H202 in methanol for 30 min at 22°C. Rehydration was performed with graded ethanol solutions (100%, 95%, 80%, 70%, 50%) and H20, each for a 3-min period. Nonspecific staining was reduced by incubation with 10% heat-inactivated normal goat seru...

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We have previously shown the development in vitro of tryptase+ human mast cells from fetal liver cells cocultured with murine 3T3 fibroblasts. In this study, recombinant human stem cell factor (rhuSCF), the ligand for the c-kit proto-oncogene product called Kit, stimulated the growth and differentiation primarily of mast cells from dispersed fetal liver cells, whereas recombinant human interleukin-3 (rhuIL-3) stimulated the differentiation of basophils along with other cell types. Cultures of fetal liver cells were initiated and maintained in the presence of rhuSCF or rhuIL-3 for up to 6 weeks. Metachromatic cells in cytospins were identified as mast cells primarily on the basis of tryptase expression, and as MCT or MCTC by immunohistochemistry using monoclonal antibodies against tryptase and chymase, whereas basophils were metachromatic, polymorphonuclear, and lacked these proteases. Levels of tryptase and histamine were measured by radioimmunoassay, tryptase and chymase activities by peptide hydrolysis, and cell surface Kit by flow cytometry with the monoclonal antibody YB5.B8. The predominant presence of mast cells occurred only in the cultures supplemented with rhuSCF. The percentage and total number of mast cells increased over time with increasing concentrations of rhuSCF and reached a plateau at 55 ng/mL. At this concentration of rhuSCF, mast cells first appeared by day 7; by day 42, 106% of the starting number of cells were present and 85% of these were tryptase+, 31% being weakly chymase+. These mast cells appeared immature by ultrastructural criteria; most cells were mononuclear, but some had nuclei with deeply divided lobes. DNA synthesis in tryptase+ mast cells at days 21 and 28 of culture with rhuSCF was demonstrated by incorporation of bromodeoxyuridine. Calculated levels of histamine (1.2 pg/mast cell) and tryptase (0.9 pg/mast cell) were similar to those determined previously in coculture experiments with murine 3T3 fibroblasts. Chymase activity was undetectable in most cell extracts. On day 0, 4% to 20% of fetal liver cells expressed cell surface Kit. In the presence of rhuSCF, the percentages and total numbers of Kit+ cells and the apparent concentration of Kit per cell increased along with the number of tryptase+ cells. In the presence of rhuIL-3, toluidine blue+, tryptase- cells first and maximally appeared at day 14 (11% +/- 2.5%). The percentage of these toluidine blue+ cells then declined to about 6% by days 21 and 35, while the total number of positive cells declined over 10-fold. Kit+ cells in the presence of rhuIL-3 declined from 9% on day 3 to 2% on day 35.(ABSTRACT TRUNCATED AT 400 WORDS)

Hematologic and serologic abnormalities similar to those observed in human systemic lupus erythematosus (SLE) developed in cynomolgus macaques fed alfalfa sprouts. L-Canavanine sulfate, a constituent of alfalfa sprouts, was incorporated into the diet and reactivated the syndrome in monkeys in which an SLE-like syndrome had previously been induced by the ingestion of alfalfa seeds or sprouts.

An inherited deficiency of a metalloendopeptidase (meprin) activity occurs in kidneys of many inbred mouse strains. To clarify whether meprin protein is present in low-activity strains and determine the distribution of meprin in kidneys of mice with high- and low-meprin activities, kidney slices were stained through the use of the indirect immunoperoxidase technique and examined by light and electron microscopy. Light microscopy at high dilutions of anti-meprin IgG confirmed the brush border localization of meprin in high-meprin activity strains and revealed no detectable cross-reactive material in low-meprin activity strains. However, light and electron microscopy studies that use lower dilutions of anti-meprin immunoglobulin G (IgG) revealed cross-reactivity in low-activity strains, also at the luminal surface of the proximal tubules. Studies at lower magnifications indicated that meprin is primarily associated with the juxtamedullary region of the kidney in both high- and low-activity strains. Western blots of urinary proteins showed significant amounts of meprin-like proteins, but only in the urine of mice with high-meprin activity. The low activity of meprin in some inbred mouse strains is not associated with the presence of the protein in compartments of kidney cells other than the brush border or with secretion of the protein into the urine.